In order to produce chondroitin sulfate in an animal-free manner, engineered E. coli host cells were modified so as to reduce expression of an endogenous gene for fructosyltransferase (kfoE); reduce expression of an endogenous gene for 3′-phosphoadenosine-5′-phosphosulfate reductase (cysH); and express one or more exogenous sulfotransferases. Expression of proteins forming ATP-binding cassette transporters were also reduced to limit export of glycosaminoglycans from the cells. The recombinant microorganisms are able produce all three components identified for chondroitin sulfate production—chondroitin, sulfate donor, and sulfotransferase. These modified E. coli are capable of complete, essentially one-step biosynthesis of chondroitin sulfate at a variety of sulfation levels from simple microbial media components and glucose. This is a major advantage over current production methods that depend on the natural distribution of chondroitin sulfate types in the animal tissue.

The present invention relates to a method for chondroitin sulfate biosynthesis, belongs to the field of pharmaceuticals. CS was biosynthesized by sulfating the chondroitin with C4ST or C6ST in Tris-HCl buffer assisted with 3′-phosphoadenosine 5′-phophosulfate (PAPS). C4ST and C46ST came from bioengineered Escherichia coli or Pichia pastoris. Chondroitin came from bioengineered Bacillus subtilis 168.

The present invention relates to a method for chondroitin sulfate biosynthesis, belongs to the field of pharmaceuticals field. CS was biosynthesized by sulfating the chondroitin with C4ST or C6ST in Tris-HCI buffer assisted with 3-phosphoadenosine 5-phophosulfate (PAPS). C4ST and C46ST came from bioengineered Escherichia coli or Pichia pastoris. Chondroitin came from bioengineered Bacillus subtilis 168.

Disclosed is a method for constructing a synthesis and regeneration system based on APS as an active sulfonate donor, belonging to the technical field of biology. The present disclosure provides a new purpose of APS as an active sulfonate donor, and greatly improves the synthesis efficiency of the APS by screening different ATP sulfurylases and adding a pyrophosphatase into a reaction system to eliminate pyrophosphate as a byproduct. Further, the construction of a sulfonation modification system is realized by constructing an APS circulation regeneration system. Compared with a PAPS regeneration system, the APS circulation regeneration system has the advantages of short path and high efficiency, the sulfonation modification efficiency is significantly improved, and the synthesis cost is successfully reduced.